Cellular Changes during Renal Failure-Induced Inflammatory Aortic Valve Disease

Background

Aortic valve calcification (AVC) secondary to renal failure (RF) is an inflammation-regulated process, but its pathogenesis remains unknown. We sought to assess the cellular processes that are involved in the early phases of aortic valve disease using a unique animal model of RF-associated AVC.

Methods

Aortic valves were obtained from rats that were fed a uremia-inducing diet exclusively for 2, 3, 4, 5, and 6 weeks as well as from controls. Pathological examination of the valves included histological characterization, von Kossa staining, and antigen expression analyses.

Results

After 2 weeks, we noted a significant increase in urea and creatinine levels, reflecting RF. RF parameters exacerbated until the Week 5 and plateaued. Whereas no histological changes or calcification was observed in the valves of any study group, macrophage accumulation became apparent as early as 2 weeks after the diet was started and rose after 3 weeks. By western blot, osteoblast markers were expressed after 2 weeks on the diet and decreased after 6 weeks. Collagen 3 was up-regulated after 3 weeks, plateauing at 4 weeks, whereas collagen 1 levels peaked at 2 and 4 weeks. Fibronectin levels increased gradually until Week 5 and decreased at 6 weeks. We observed early activation of the ERK pathway, whereas other pathways remained unchanged.

Conclusions

We concluded that RF induces dramatic changes at the cellular level, including macrophage accumulation, activation of cell signaling pathway and extracellular matrix modification. These changes precede valve calcification and may increase propensity for calcification, and have to be investigated further.     

PEP-1-FK506BP12 inhibits matrix metalloproteinase expression in human articular chondrocytes and in a mouse carrageenan-induced arthritis model

The 12 kDa FK506-binding protein (FK506BP12), an immunosuppressor, modulates T cell activation via calcineurin inhibition. In this study, we investigated the ability of PEP-1-FK506BP12, consisting of FK506BP12 fused to the protein transduction domain PEP-1 peptide, to suppress catabolic responses in primary human chondrocytes and in a mouse carrageenan-induced paw arthritis model. Western blotting and immunofluorescence analysis showed that PEP-1-FK506BP12 efficiently penetrated chondrocytes and cartilage explants. In interleukin-1β (IL-1β)-treated chondrocytes, PEP-1-FK506BP12 significantly suppressed the expression of catabolic enzymes, including matrix metalloproteinases (MMPs)-1, -3, and -13 in addition to cyclooxygenase-2, at both the mRNA and protein levels, whereas FK506BP12 alone did not. In addition, PEP-1-FK506BP12 decreased IL-1β-induced phosphorylation of the mitogen-activated protein kinase (MAPK) complex (p38, JNK, and ERK) and the inhibitor kappa B alpha. In the mouse model of carrageenan-induced paw arthritis, PEP-1-FK506BP12 suppressed both carrageenan-induced MMP-13 production and paw inflammation. PEP-1-FK506BP12 may have therapeutic potential in the alleviation of OA progression. [BMB Reports 2015; 48(7): 407-412]     

Association of Erythropoietin-Stimulating Agent Responsiveness with Mortality in Hemodialysis and Peritoneal Dialysis Patients

Erythropoiesis-stimulating agent (ESA) responsiveness has been reported to be associated with increased mortality in hemodialysis (HD) patients. ESA requirement to obtain the same hemoglobin (Hb) level is different between HD and peritoneal dialysis (PD) patients. In this study, we investigated the impact of ESA responsiveness on mortality between both HD and PD patients. Prevalent HD and PD patients were selected from the Clinical Research Center registry for end-stage renal disease, a prospective cohort study in Korea. ESA responsiveness was estimated using an erythropoietin resistant index (ERI) (U/kg/week/g/dL). Patients were divided into three groups by tertiles of ERI. ESA responsiveness was also assessed based on a combination of ESA dosage and hemoglobin (Hb) levels. The primary outcome was all-cause mortality. A total of 1,594 HD and 876 PD patients were included. The median ESA dose and ERI were lower in PD patients compared with HD patients (ESA dose: 4000 U/week vs 6000 U/week, respectively. P<0.001, ERI: 7.0 vs 10.4 U/kg/week/g/dl, respectively. P<0.001). The median follow-up period was 40 months. In HD patients, the highest ERI tertile was significantly associated with higher risk for all-cause mortality (HR 1.96, 95% CI, 1.07 to 3.59, P = 0.029). HD patients with high-dose ESA and low Hb levels (ESA hypo-responsiveness) had a significantly higher risk of all-cause mortality (HR 2.24, 95% CI, 1.16 to 4.31, P = 0.016). In PD patients, there was no significant difference in all-cause mortality among the ERI groups (P = 0.247, log-rank test). ESA hypo-responsiveness was not associated with all-cause mortality (HR = 1.75, 95% CI, 0.58 to 5.28, P = 0.319). Our data showed that ESA hypo-responsiveness was associated with an increased risk of all-cause mortality in HD patients. However, in PD patients, ESA hypo-responsiveness was not related to all-cause mortality. These finding suggest the different prognostic value of ESA responsiveness between HD and PD patients.     

Sex-Related Online Behaviors,Perceived Peer Norms and Adolescents’ Experience with Sexual Behavior: Testing an Integrative Model

Research on the role of sex-related Internet use in adolescents’ sexual development has often isolated the Internet and online behaviors from other, offline influencing factors in adolescents’ lives, such as processes in the peer domain. The aim of this study was to test an integrative model explaining how receptive (i.e., use of sexually explicit Internet material [SEIM]) and interactive (i.e., use of social networking sites [SNS]) sex-related online behaviors interrelate with perceived peer norms in predicting adolescents’ experience with sexual behavior. Structural equation modeling on longitudinal data from 1,132 Dutch adolescents (M_age T₁ = 13.95; range 11-17; 52.7% boys) demonstrated concurrent, direct, and indirect effects between sex-related online behaviors, perceived peer norms, and experience with sexual behavior. SEIM use (among boys) and SNS use (among boys and girls) predicted increases in adolescents’ perceptions of peer approval of sexual behavior and/or in their estimates of the numbers of sexually active peers. These perceptions, in turn, predicted increases in adolescents’ level of experience with sexual behavior at the end of the study. Boys’ SNS use also directly predicted increased levels of experience with sexual behavior. These findings highlight the need for multisystemic research and intervention development to promote adolescents’ sexual health.     

Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4⁺ T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.     

MT2 Melatonin Receptor Immunoreactivity in Neurons is Very High in the Aged Hippocampal Formation in Gerbils

Melatonin exerts many physiological functions via its G protein-coupled receptors. In the present study, we investigated age-related changes in MT2 melatonin receptor immunoreactivity and its levels in the gerbil hippocampus during normal aging. In the postnatal month 1 (PM 1) group, MT2 immunoreaction was well observed in neurons in all subregions of the gerbil hippocampus. In the PM 3 and 6 groups, MT2 immunoreactivity in neurons was decreased compared to that in the PM 1 group. Thereafter, MT2 immunoreactivity in neurons was increased. In the PM 18 and 24 groups, MT2 immunoreactivity in neurons was strong in all subregions of the gerbil hippocampus. In addition, the number of MT2 immunoreactive cells was lowest at PM 3 and highest at PM 24. From western blot analysis, age-dependent change pattern in MT2 level in the gerbil hippocampus was similar to the immunohistochemical result. These results indicate that MT2 immunoreactivity and levels are altered in the gerbil hippocampus during normal aging; lowest at young adult stage and highest at aged stage.     

Development and organization of polarity-specific segregation of primary vestibular afferent fibers in mice

A striking feature of vestibular hair cells is the polarized arrangement of their stereocilia as the basis for their directional sensitivity. In mammals, each of the vestibular end organs is characterized by a distinct distribution of these polarized cells. We utilized the technique of post-fixation transganglionic neuronal tracing with fluorescent lipid soluble dyes in embryonic and postnatal mice to investigate whether these polarity characteristics correlate with the pattern of connections between the endorgans and their central targets; the vestibular nuclei and cerebellum. We found that the cerebellar and brainstem projections develop independently from each other and have a non-overlapping distribution of neurons and afferents from E11.5 on. In addition, we show that the vestibular fibers projecting to the cerebellum originate preferentially from the lateral half of the utricular macula and the medial half of the saccular macula. In contrast, the brainstem vestibular afferents originate primarily from the medial half of the utricular macula and the lateral half of the saccular macula. This indicates that the line of hair cell polarity reversal within the striola region segregates almost mutually exclusive central projections. A possible interpretation of this feature is that this macular organization provides an inhibitory side-loop through the cerebellum to produce synergistic tuning effects in the vestibular nuclei. The canal cristae project to the brainstem vestibular nuclei and cerebellum, but the projection to the vestibulocerebellum originates preferentially from the superior half of each of the cristae. The reason for this pattern is not clear, but it may compensate for unequal activation of crista hair cells or may be an evolutionary atavism reflecting a different polarity organization in ancestral vertebrate ears.     

Phosphorylated extracellular signal-regulated kinase 1/2 immunoreactivity and its protein levels in the gerbil hippocampus during normal aging

Phosphorylated extracellular signal-regulated kinase (pERK) mediates neuronal synaptic plasticity, long-term potentiation, and learning and memory in the hippocampus. In this study, we examined pERK1/2 immunoreactivity and its protein level in the gerbil hippocampus at various ages. In the postnatal month 1 (PM 1) group, very weak pERK1/2 immunoreactivity was detected in the hippocampus. In the CA1 region, pERK1/2 immunoreactivity was considerably increased in the stratum pyramidale in the PM 6 group. Thereafter, pERK1/2 immunoreactivity was decreased. In the CA2/3 region, pERK1/2 immunoreactivity increased in an age-dependent manner until PM 12. Thereafter, numbers of pERK1/2-immunoreactive neurons were decreased. However, in the mossy fiber zone, pERK1/2 immunostaining became stronger with age. In the dentate gyrus, a few pERK1/2-immunoreactive cells were observed until PM 12. In the PM 18 and 24 groups, numbers of pERK1/2-immunoreactive cells were increased, especially in the polymorphic layer. In Western blot analysis, pERK1/2 level in the gerbil hippocampus was increased with age. These results indicate that total pERK1/2 levels are increased in the hippocampus with age. However pERK1/2 immunoreactivity in subregions of the gerbil hippocampus was changed with different pattern during normal aging.     

Cloning,large-scale production,and purification of active dimeric rat vascular endothelial growth factor (rrVEGF-164)

Large-scale production of recombinant rat vascular endothelial growth factor (rrVEGF-164) is desirable for angiogenic studies. In this study, biologically active recombinant rat vascular endothelial growth factor (rrVEGF-164) was cloned and expressed in the yeast Pichia pastoris, and large-scale production was performed by fermentation. cDNA encoding VEGF-164 was prepared from embryonic rat tissue RNA, and a recombinant pPIC9HV/rVEGF-164 plasmid, containing an AOX1 promoter, was constructed. The methylotrophic P. pastoris was used as the eukaryotic expression system. After transformation, rrVEGF-164 was produced by fermentation (～124 mg/L) and purified by heparin affinity chromatography. SDS–PAGE indicated that rrVEGF-164 was produced as a disulphide-bridged dimer of 48 kDa which was purified to near homogeneity by heparin affinity chromatography in a large quantity. A bioassay indicated a three- to fivefold increase in endothelial cell proliferation after 3 days, due to the addition of the produced rrVEGF-164. The produced rrVEGF-164 showed a higher biological activity than a commercially available, mouse cell line-based, growth factor. In conclusion, using the P. pastoris expression system we were able to produce biologically active rat VEGF-164 in high quantities and this may provide a powerful tool for basic and applied life sciences.     

Intrastriatal grafts of mesenchymal stem cells in adult intact rats can elevate tyrosine hydroxylase expression and dopamine levels

MSCs (mesenchymal stem cells) derived from the bone marrow have shown to be a promising source of stem cells in a therapeutic strategy of neurodegenerative disorder. Also, MSCs can enhance the TH (tyrosine hydroxylase) expression and DA (dopamine) content in catecholaminergic cells by in vitro co‐culture system. In the present study, we investigated the effect of intrastriatal grafts of MSCs on TH protein and gene levels and DA content in adult intact rats. When MSCs were transplanted into the striatum of normal rats, the grafted striatum not only had significantly higher TH protein and mRNA levels, but also significantly higher DA content than the untransplanted striatum. Meanwhile, the grafted MSCs differentiated into neurons, astrocytes and oligodendrocytes; however, TH‐positive cells could not be detected in our study. These experimental results offer further evidence that MSCs are a promising candidate for treating neurodegenerative diseases such as Parkinson's disease.